Numerous types of RNAs are essential to all forms of life. They constitute the genomes of viruses, act as catalysts and as modifiers of gene expression, and translate the flow of genetic information into protein. In all of these roles, RNA molecules must be specifically recognized by the cognate partners to correctly perform their biological functions. The recognition of tRNAs by aminoacyl-tRNA synthetase enzymes serves as a paradigm to unravel recognition mechanisms. This project uses genetics, biochemistry and three-dimensional structural determinations in converging operations to define tRNA recognition specificity in molecular detail. The work is driven by in vivo mutant selections of redesigned tRNAs that retain specificity in tRNA- gene-deleted strains of Escherichia coli. In close collaborations with structural biologists in England and in France, the functions of mutant tRNAs are interpreted in terms of their molecular structures in the enzyme complex. The work has wide implications because the dissection of simple RNA recognition tags will identify the features that allow their use throughout biology. In addition, the species-specific variations in tRNAs and aminoacyl-tRNA synthetases makes them potential targets for therapeutic agents. During the next grant period, the specific aims are: 1. To improve the understanding of the features that mark the G-U wobble pair in alanine tRNA for recognition. 2. To delineate features in aspartic acid tRNA that insure its recognition specificity in the cellular setting where twenty systems operate simultaneously. 3. To explore the mechanisms through which distal contacts activate the enzyme catalytic center in the arginine system. 4. To examine the role of G-U wobble pairs in promoting tRNA flexibility as the molecule moves through the protein synthesis machinery.